Data_Sheet_1_Photoperiod and Vernalization Control of Flowering-Related Genes: A Case Study of the Narrow-Leafed Lupin (Lupinus angustifolius L.).XLSX

Narrow-leafed lupin (Lupinus angustifolius L.) is a moderate-yielding legume crop known for its high grain protein content and contribution to soil improvement. It is cultivated under photoperiods ranging from 9 to 17 h, as a spring-sown (in colder locations) or as an autumn-sown crop (in warmer regions). Wild populations require a prolonged cold period, called vernalization, to induce flowering. The key achievement of L. angustifolius domestication was the discovery of two natural mutations (named Ku and Jul) conferring vernalization independence. These mutations are overlapping deletion variants in the promoter of LanFTc1, a homolog of the Arabidopsis thaliana FLOWERING LOCUS T (FT) gene. The third deletion, named here as Pal, was recently found in primitive germplasm. In this study, we genotyped L. angustifolius germplasm that differs in domestication status and geographical origin for LanFTc1 alleles, which we then phenotyped to establish flowering time and vernalization responsiveness. The Ku and Jul lines were vernalization-independent and early flowering, wild (ku) lines were vernalization-dependent and late flowering, whereas the Pal line conferred intermediate phenotype. Three lines representing ku, Pal, and Ku alleles were subjected to gene expression surveys under 8- and 16-h photoperiods. FT homologs (LanFTa1, LanFTa2, LanFTc1, and LanFTc2) and some genes selected by recent expression quantitative trait loci mapping were analyzed. Expression profiles of LanFTc1 and LanAGL8 (AGAMOUS-like 8) matched observed differences in flowering time between genotypes, highlighted by high induction after vernalization in the ku line. Moreover, these genes revealed altered circadian clock control in Pal line under short days. LanFD (FD) and LanCRLK1 (CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1) were negatively responsive to vernalization in Ku and Pal lines but positively responsive or variable in ku, whereas LanUGT85A2 (UDP-GLUCOSYL TRANSFERASE 85A2) was significantly suppressed by vernalization in all lines. Such a pattern suggests the opposite regulation of these gene pairs in the vernalization pathway. LanCRLK1 and LanUGT85A2 are homologs of A. thaliana genes involved in the FLOWERING LOCUS C (FLC) vernalization pathway. Lupins, like many other legumes, do not have any FLC homologs. Therefore, candidate genes surveyed in this study, namely LanFTc1, LanAGL8, LanCRLK1, and LanUGT85A2, may constitute anchors for further elucidation of molecular components contributing to vernalization response in legumes.

窄叶羽扇豆（Lupinus angustifolius L.）是一种中等产量的豆科作物，以其高籽粒蛋白含量及土壤改良价值而广受认可。该作物的种植光周期范围为9至17小时，在寒冷地区多采用春播模式，温暖地区则以秋播为主。野生窄叶羽扇豆种群需要经历持续低温处理（即春化作用（vernalization））才能诱导开花。窄叶羽扇豆驯化历程中的关键突破，是发现了两个可赋予植株春化不依赖特性的自然突变体（分别命名为Ku和Jul）。上述突变均为LanFTc1启动子区域的重叠缺失变异，而LanFTc1是拟南芥（Arabidopsis thaliana）开花位点T（FLOWERING LOCUS T，FT）基因的同源基因。近期在原始种质中还发现了第三个缺失变体，本研究将其命名为Pal。本研究对具有不同驯化状态和地理起源的窄叶羽扇豆种质开展了LanFTc1等位基因分型，随后对其进行表型分析以明确开花时间与春化响应特性。结果显示，携带Ku和Jul等位基因的品系无春化依赖特性且开花较早；野生型（ku）品系依赖春化且开花较晚；而携带Pal等位基因的品系则表现出中间型表型。本研究选取分别携带ku、Pal及Ku等位基因的三个品系，在8小时和16小时光周期条件下进行基因表达检测。分析对象包括FT同源基因（LanFTa1、LanFTa2、LanFTc1和LanFTc2），以及近期通过表达数量性状位点（expression quantitative trait loci，eQTL）作图筛选得到的部分基因。LanFTc1与LanAGL8（AGAMOUS-like 8，AGAMOUS同源盒8）的表达谱与各基因型间的开花时间差异高度匹配，其中ku品系经春化处理后这两个基因的表达量显著上调。此外，Pal品系在短日照条件下呈现出昼夜节律调控异常的特征。LanFD（FD，成花素互作蛋白FD）与LanCRLK1（CALCIUM/CALMODULIN-REGULATED RECEPTOR-LIKE KINASE 1，钙/钙调素调控受体样激酶1）在Ku和Pal品系中对春化处理呈负响应，而在ku品系中则呈正响应或响应不稳定；LanUGT85A2（UDP-GLUCOSYL TRANSFERASE 85A2，UDP-糖基转移酶85A2）在所有品系中均因春化处理而显著下调。这种表达模式表明，这两对基因在春化通路中存在相反的调控机制。LanCRLK1与LanUGT85A2是拟南芥中参与开花位点C（FLOWERING LOCUS C，FLC）春化通路的基因的同源基因。与多数其他豆科植物类似，羽扇豆并未发现FLC同源基因。因此，本研究中检测的候选基因LanFTc1、LanAGL8、LanCRLK1及LanUGT85A2，可作为进一步解析豆科植物春化响应分子组分的研究锚点。